ann. anim. sci., vol. 21, no. 4 (2021) 1435–1454 doi: 10

Ann. Anim. Sci., Vol. 21, No. 4 (2021) 1435–1454 DOI: 10.2478/aoas-2021-0011

The effecT of adding molasses in differenT Times on performance of nile Tilapia (OreOchrOmis nilOticus)

raised in a low-saliniTy biofloc sysTem

Mohammad Hossein Khanjani1♦, Morteza Alizadeh2, Mohammad Mohammadi2, Habib Sarsangi Aliabad2

1Department of Fisheries Sciences and Engineering, Faculty of Natural Resources, University of Jiroft, Jiroft, Iran

2Saline Water Aquatic National Research Center, Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research, Education and Extension Organization (AREEO), Bafgh, Iran

♦Corresponding author: [email protected], [email protected]

abstractThis study aimed to evaluate the effect of adding molasses in different times on water quality, growth performance, body biochemical composition, digestive and hepatic enzymes of nile tila-pia in the biofloc system. Tilapia fingerlings (1.53 ± 0.14 g) were distributed in five treatments including control, BFT24 (adding molasses to the tanks every 24 h), BFT48 (48 h), BFT72 (72 h), and BFT96 (96 h) and reared for 37 days in fiberglass tanks (130 L), with a stocking density of one fish per litre. The results showed that highest increases in biomass (740.12 g) and survival (98.97%) were obtained in BFT24 treatment (P<0.05). The body composition was affected by the experimental treatments so that the highest protein content was obtained in the BFT72 (P<0.05). Digestive enzymes activities were significantly (P<0.05) higher in BFT treatments than the control group. The current study showed higher biomass and survival ratio for Nile tilapia were observed in BFT24 treatment. The liver and digestive enzymes of Nile tilapia were affected by the different addition times of molasses to the rearing tanks.

Key words: Nile tilapia, biofloc, molasses, growth performance, liver and digestive enzymes

The increasing development of the aquaculture industry as one of the leading sources of human food supply has led to the adoption of advanced and environmen-tally friendly technologies. In recent years, biofloc technology has been recognized as an efficient system in the aquaculture industry (Khanjani and Sharifinia, 2020). Environmental compatibility (Emerenciano et al., 2017; Khanjani and Sharifinia, 2020), reducing the need for natural resources (García-Ríos et al., 2019; Avnime-lech, 2012), increasing the stocking density of cultured species (Lima et al., 2018)

M.H. Khanjani et al.1436

and controlling nitrogenous waste (Emerenciano et al., 2017) are mentioned as fea-tures of the biofloc system. Bioflocs are composed of abiotic (food debris, feces, and organic particles) and biotic (heterotrophic and chemoautotrophic bacteria) compo-nents (Khanjani and Sharifinia, 2020) that are available 24 hours a day as a rich food source for filter feeder aquatic species and improve fish production (Hu et al., 2015). Various studies have examined biofloc as a source of protein supplement in the diet (Xu and Pan, 2014; Khatoon et al., 2016; Khanjani et al., 2020 a, b).

Nile tilapia (Oreochromis niloticus) is one of the main species that has gained worldwide popularity due to the white meat with a firm texture and the absence of spines within the muscle (Wang and Lu, 2016). This species is fast-growing (El-Sayed, 2006), resistant to various environmental conditions, adapted to high stock-ing densities (Avnimelech, 2007) and the second-largest freshwater species grown in the world (Menaga et al., 2019). Moreover, tilapia species is one of the most suit-able and compatible species in the biofloc system and its potential has been inves-tigated in various studies. Cultivation of tilapia species in biofloc systems has been examined from different aspects: effects of various stocking densities (Haridas et al., 2017; Lima et al., 2018), the performance of growing period and overwintering (Crab et al., 2009), the larval stage (Ekasari et al., 2015), fingerling period (García-Ríos et al., 2019), broodstocks (Ekasari et al., 2015), carbon to nitrogen ratio and dif-ferent carbon sources (Pérez-Fuentes et al., 2016; Mirzakhani et al., 2019; Khanjani et al., 2021 a), combining with the aquaponic system (Pinho et al., 2017), cultivation in brackish water under the influence of protein and digestible energy (Durigon et al., 2020).

Tilapia is a brilliant group of fish for cultivation in freshwater and brackish water since they are tolerant of salinity between 0 and 12 g L-1 (El-Sayed, 2006). Moreover, freshwater fish cultivated in brackish water can save energy leading to higher growth rate (Lima et al., 2018). Increased salinity may also reduce the toxicity of nitrogen compounds (Colt, 2006).

The biofloc in the production system affects water quality (García-Ríos et al., 2019; Ren et al., 2019), growth performance (Khanjani et al., 2020 b; Durigon et al., 2020), digestive enzyme activity (Najdegerami et al., 2016; Xu and Pan, 2012), immunity (Long et al., 2015; Menaga et al., 2019), antioxidant status (Bakhshi et al., 2018; Liu et al., 2018) and body composition of the cultured species (Mirzakhani et al., 2019; Khanjani et al., 2020 a).

Microbial communities in the biofloc systems act like probiotics (Aguilera-Ri-vera et al., 2014) and positively affect digestive and hepatic enzymes (Adorian et al., 2019). Different conditions include temperature (Apún-Molina et al., 2009), pH (Márquez et al., 2012), feeding habits (Seixas Filho et al., 2000), diet composition (Durigon et al., 2019; Hakim et al., 2006), nitrogen conversion pathway (hetero-trophic or autotrophic) (Becerra-Dórame et al., 2012) and production system as cage or pond (Santos et al., 2016) affects growth performance and digestive enzymes’ activity in cultured species.

Various studies have been done in the context of adding different carbon sources to the biofloc system (Pérez-Fuentes et al., 2016; Deng et al., 2018; Mirzakhani et al., 2019; Khanjani et al., 2021 a). Molasses as an organic carbon source is successfully

Effect of molasses on Nile tilapia raised in a biofloc system 1437

used in the biofloc system for growth of heterotrophic bacteria in tilapia cultivation ponds (Khanjani et al., 2021 a, b).

It is important to determine the most appropriate time to add carbon to the bio-floc system and better system management, in terms of environmental issues and development of sustainable aquaculture. There are no studies to date on the influence of adding carbonated organic matters (molasses) in different times to the system with limited water exchange. Therefore, the present study was conducted to evaluate the effects of adding molasses in different times on water quality, growth performance, somatic indices, liver and digestive enzymes, and body biochemical composition of Nile tilapia in the biofloc system with limited water exchange.

material and methods

experimental designNile tilapia fingerlings with an average weight of 1.53 ± 0.14 g and length of

3.98 ± 0.1 mm (Mean ± SD) were obtained from National Research Center of Saline Water Aquatics (Bafgh city, Yazd province, Iran). The experiment was performed at the mentioned research center. Altogether, 15 tanks (with a total volume of 300 liters) were considered for this experiment. Each tank was filled with 130 liters of well wa-ter at a salinity of 8 ppt, and then 130 fingerlings of Nile tilapia (1.53 g L–1 biomass) were stocked in each tank. The experimental period was 37 days.

Five experimental treatments were considered for the present study, including a water exchange treatment (control group) where 50 to 60% of the water in the rear-ing tank was replaced daily with fresh water with the same salinity before feeding; and other four biofloc treatments by adding carbonaceous molasses to the cultivation tanks (adding molasses to the tanks was done 24 h as BFT24, 48 h as BFT48, 72 h as BFT72, and 96 h as BFT96) with 0.4 to 0.8% daily water exchange from the bottom of the tanks.

In the biofloc treatments, 2.5 mL biofloc per liter was added to the tanks as the initial stock (Khanjani et al., 2021 a). The initial stock was collected from three growing ponds of Nile tilapia (6 m in diameter and 1.2 m in height) based on biofloc. Feeding was performed three times a day (08:00, 12:00 and 16:00) with a diet con-taining 35% protein, 9% lipid, 4% fibre, 12% ash, C/N ratio 8.9 (manufactured by Mazandaran Animal and Aquatic Feed Company, MANAQUA, Iran) and based on body weight. At the beginning of the trial, the feeding rate was considered 8% and the rate of feeding decreased with an increase in the growth. In biofloc treatments, feeding was 25% lower than control. The fish were fed in biofloc treatments with 25% of the produced flocs and 75% of the artificial diet.

For aeration and oxygen supply, three air stones were placed at the tanks that were connected to the aerator. Aeration was performed continuously throughout the day and night to supply oxygen. In addition, it causes microbial flocs to remain sus-pended and be made available to fish as a complementary food.

The amount of molasses (dry matter: 55.18%, crude protein: 9.16%, lipid: 0.36%, fiber: 0.88%, ash: 16.36% and carbohydrate: 73.24%) was calculated based


on the amount of feed and the amount of nitrogen which is released into the water, which was approximately 0.6 g of feed for the carbon to nitrogen ratio of 15. It was added to the biofloc treatments after noon meal according to Avnimelech (2009). The amount of carbonaceous material added to the system was considered equal in all treatments. After weighing, the molasses was poured into a one-liter plastic container and mixed well with the water of the growing tanks and spread evenly across the tank surface (In various treatments: as per their treatment times).

physicochemical parametersWater quality factors including temperature, pH and dissolved oxygen were

measured twice daily at 8 am and 16 pm. Salinity was determined daily at 9 o’clock using HQ30D Multi Meter HACH. Transparency, settled solids (SS), and total sus-pended solids (TSS) were measured every after five days’ interval.

Transparency was determined with a Secchi disk. To determine the amount of settled solid, one liter of rearing water was poured into Imhoff funnel (scaled conical hopper) and held for 20 minutes to settle (Avnimelech and Kochba, 2009).

To measure total suspended solids, Whatman filter paper 42 (No 1442) was first numbered and weighted for each treatment and then 100 ml of tank water was fil-tered and placed in the oven at 105°C for 3 hours to dry. Then, it was placed in a desiccator to absorb moisture, and finally, its weight difference was calculated (Khanjani et al., 2017).

Total counts of heterotrophic bacteria (CFU: colony-forming units) were per-formed using culture medium (R2-agar) according to the APHA (2005) method. To-tal ammonium nitrogen (TAN), nitrite and nitrate levels were measured weekly with a spectrophotometer (Perkin Elmer Lambda 25 UV/Vis) according to MOOPAM (1999).

measurement of growth, nutritional and somatic indicesTwenty fish per each replicate were used to measure growth, nutritional and

somatic indices. Fish biometrics (length and weight) were done at the beginning and end of the experiment to calculate growth indices including weight gain (WG), body weight index (BWI), daily weight gain (DWG), biomass and specific growth rate (SGR). The number of fish at the beginning and the end of the experiment were recorded to calculate the survival rate (SR). Nutrition indices including feed conver-sion ratio (FCR), feed efficiency (FE) and protein efficiency ratio (PER) were calcu-lated based on the following equations (Khanjani et al., 2016):

WG (g) = final fish weight – initial fish weight,BWI (%) = WG (g)/initial fish weight (g) × 100, DWG (g) = WG (g)/days of rearing,Biomass (g) = WG (g) × SR (%) × initial number of fish,SGR (%/day) = [(Ln (final fish weight) – Ln (initial fish weight) ×100]/days of

rearing,SR (%) = (number of individuals at end of rearing period/initial number of indi-

viduals stocked) × 100,FCR = feed consumed (g)/WG (g),


FE (%) = WG (g)/ feed consumed (g) ×100,PER= WG (g)/protein intake (g).Somatic indices including condition factor (CF), viscerosomatic index (VSI),

hepatosomatic index (HSI), carcass yield (CY) were also determined based on the following equations (Durigon et al., 2020):

Condition factor, CF = 100 × total weight (g)/body length3 (cm),Viscerosomatic index (%), VSI =100 × visceral weight (g)/final fish weight (g),Hepatosomatic index (%), HIS =100 × liver weight (g)/final fish weight (g),Carcass yield (%), CY = 100 × total fish weight – visceral weight (g)/total fish

weight (g).

proximate analysis of the carcass biochemical compositionTwenty fish from each replicate were randomly separated from the tanks at the

end of the experiment. They were put in the ice until they became numb (ethical issues were followed to kill the fish). Then, the contents of the abdomen were com-pletely removed and the rest of the body was homogenized with the meat grinder. After that, the samples were stored at –18°C until the analysis. Subsequently, protein, lipid, moisture, and ash were measured using the AOAC (2005) method.

Biofloc analysisAt the end of the experimental period, the water of each treatment was passed

through 20 μm nets, and the produced biofloc from each treatment was placed in pre-numbered containers. Then, the samples were kept in an oven at 72°C for 72 h until dried. After that, the dried bioflocs were stored in the freezer at –18°C until biochemical analysis. The chemical factors of protein, lipid, moisture, and ash were measured based on AOAC (2005) method.

digestive enzyme activityAt the end of the experiment, the feeding was cut off 24 h before sampling. Five

fish were randomly removed from each replicate and anesthetized with clove powder (200 ppm) and killed by observing ethical issues.

The intestine was removed by abdominal opening and visceral fat was placed on ice. Tissues were washed by cold normal saline solution and stored at –80ºC. In-testine tissue was thawed and homogenized in 1:5 (w/v) cold 50 mM tris-HCl buffer, pH=7.5 for enzyme extract preparation. The homogenates were centrifuged (10,000 g for 20 min at 4ºC) and the resultant supernatant was separated and frozen at –80ºC until analyses.

Alkaline protease activity was measured according to Garcia-Carreno and Haard (1993). Azocasein 2% in Tris-HCl, pH= 7.5 was applied as the substrate. The lipase specific activity was determined by the method described by Iijima et al. (1998); in this method nitrophenyl myristate was used as the substrate. Amylase activity was measured according to Bernfeld (1955); in this measurement starch was used as the substrate. The activity of all enzymes was expressed as the specific activity being micromole of substrate hydrolyzed per minute per milligram protein (u/mg) (Jenabi Haghparast et al., 2019).


hepatic enzyme activityAt the end of the experiment, 5 fish from each replicate were randomly selected,

and then anesthetized. Subsequently, they were killed by observing ethical issues. Livers were removed and kept at –80ºC. At the time of assays, liver samples were mashed on ice using porcelain mortar and pestle. Then, the samples were homog-enized in phosphate buffer (0.050 M, pH 7.4) to prepare a 10% (w/v) liver homogen-ate. Next, the homogenates were centrifuged (4ºC in 12000 rpm for 15 min) and after that, the supernatant was separated and kept at –80ºC.

Measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST) were done according to the IFCC (International Federation of Clinical Chem-istry) method (Bergmeyer et al., 1986). Alkaline phosphatase (ALP) was measured using the DGKS, 1972 (Deutsche Gesellschaft für Klinische Chemie) method. These enzymes were measured according to the photometric method by medical diagnosis kits of Pars Azmoon Company (Iran, Tehran).

statistical analysisAll data were presented as means ± standard deviation. Before analysis, the data

were checked for normality and variances for homogeneity by Shapiro-Wilk and Levene’s tests, respectively. The data were analyzed by one-way analysis of variance using the statistical software program SPSS version 21 (Chicago, Illinois, USA). Duncan’s multiple range test (DMRT) was used to determine the differences among treatments at a significance level of P<0.05. All graphs were done with Excel version 2013 (Microsoft Corporation, Redmond, WA, USA).

results

water qualityWater quality parameters during the experiment are presented in Table 1.

The amount of DO, pH, salinity, amount of SS, TSS and transparency were sig-nificantly different among treatments. The highest level of DO (7.1 mg L–1) was obtained in the control group (P<0.05). The lowest level of DO (5.92 mg L–1) and pH (7.28) were measured in the treatment which molasses was added daily (BFT24) (P<0.05).

The lowest levels of salinity (8.59 ppt) and TSS (73.59 mg L–1) were obtained in the control group. There was no significant difference in salinity, TSS, and transpar-ency among BFT treatments.

TAN and nitrate showed a higher level in BFT treatments compared to the con-trol group. The lowest levels of TAN (1.59) and NO2 (7.78 mg L–1) were obtained in the control group (P<0.05).

The fluctuations in TAN, NO2, and NO3 during the experimental period are represented in Figure 1. The average numbers of heterotrophic bacteria in different treatments are given in Table 2. The lowest (4.28 log CFU/ml) number was obtained in the control group (P<0.05).


Tabl

e 1.

Wat

er q

ualit

y pa

ram

eter

s in

tank

s of N

ile ti

lapi

a cu

ltiva

ted

unde

r the

effe

ct o

f add

ing

mol

asse

s in

diffe

rent

tim

es (M

ean

± SD

)

Para

met

ers

Con

trol

BFT

24B

FT48

BFT

72B

FT96

T a.

m. (

°C)

25.2

4±0.

31 a

25

.39±

0.34

a25

.40±

0.76

a25

.4±0

.52

a25

.5±0

.76

a

T p.

m. (

°C)

25.9

3±0.

49 a

26.1

0±0.

66 a

26.1

1±0.

73 a

26.1

4±0.

64 a

26.2

1±0.

72 a

DO

a.m

. (m

g L–1

)7.

10±0

.21

a6.

48±0

.32

c6.

81±0

.40

b6.

93±0

.33

b6.

92±0

.35

b

DO

p.m

. (m

g L–1

)6.

90±0

.146

a5.

92±0

.42

c6.

32±0

.40

b6.

29±0

.39

b6.

25±0

.48

b

pH a

.m.

7.46

5±0.

015

c7.

49±0

.016

b7.

53±0

.061

a7.

526±

0.04

a7.

53±0

.053

a

pH p

.m.

7.39

±0.0

17 c

7.28

±0.0

58 e

7.31

±0.0

47 d

7.43

±0.0

49 b

7.46

±0.0

48 a

Salin

ity (p

pt)

8.59

±0.0

7 b

8.89

±0.1

9 a

8.86

±0.2

a8.

84±0

.18

a8.

83±0

.18

a

SS (m

l L–1

)1.

53±0

.72

b20

.75±

11.7

a15

.11±

7.91

a19

.88±

12.3

a21

.4±1

5.31

a

TSS

(mg

L–1)

73.5

9±31

.67

b28

1.45

±149

.97

a22

2.93

±104

.59

a25

1.91

±143

.78

a24

0.25

±157

.14

a

Tran

spar

ency

(cm

)25

.77±

1.76

a11

.32±

6.34

b12

.91±

6.88

b12

.61±

7.5

6 b

12.3

1±7.

49 b

TAN

(mg

L–1)

1.59

±0.4

8 b

3.83

±1.1

8 a

4.76

±1.3

7 a

5.28

±1.0

1 a

5.53

±1.7

7 a

NO

2 (m

g L–1

)0.

31±0

.17

a0.

30±0

.14

a0.

22±0

.10

a0.

27±0

.13

a0.

27±0

.10

a

NO

3 (m

g L–1

)7.

78±2

.23

b18

.64±

6.64

a17

.63±

5.23

a15

.97±

4.81

a15

.95±

4.56

a

a.m

. – b

efor

e m

idda

y; p

.m. –

afte

r mid

day.

Valu

es in

the

sam

e ro

w w

ith d

iffer

ent l

ette

rs a

re si

gnifi

cant

ly d

iffer

ent (

P<0.

05).

Tem

pera

ture

= T

, Dis

solv

ed o

xyge

n =

DO

, Set

tled

solid

s = S

S, T

otal

susp

ende

d so

lids =

TSS

, Tot

al a

mm

oniu

m n

itrog

en =

TA

N, N

itrite

= N

O2,

Nitr

ate

= N

O3.

Tabl

e 2.

Tot

al d

ensi

ty o

f het

erot

roph

ic b

acte

ria (l

og c

fu/m

l) in

diff

eren

t tre

atm

ents

(Mea

n ±

SD)

BFT

96B

FT72

BFT

48B

FT24

Con

trol

5.99

± 0.

12 b

6.16

± 0

.60

ab6.

47 ±

0.5

5 ab

6.77

± 0

.55

a4.

28 ±

0.3

0 c

THB

Tota

l het

erot

roph

ic b

acte

ria: T

HB

.Va

lues

in th

e sa

me

row

with

diff

eren

t let

ters

are

sign

ifica

ntly

diff

eren

t (P<

0.05

).


Tabl

e 3.

Gro

wth

per

form

ance

and

som

atic

indi

ces o

f Nile

tila

pia

finge

rling

s in

diffe

rent

trea

tmen

ts (M

ean

± SD

)

Para

met

ers

Con

trol

BFT

24B

FT48

BFT

72B

FT96

Fina

l wei

ght (

g)7.

17±0

.74

a7.

28±0

.44

a7.

03±0

.71

a6.

37±0

.66

b6.

46±0

.69

b

Wei

ght g

ain

(g)

5.64

±0.7

4 a

5.75

±0.4

4 a

5.5±

0.71

a4.

84±0

.66

b4.

93±0

.68

b

BW

I (%

)36

8.83

±59.

59 a

375.

92±2

9.16

a35

9.63

±46.

38 a

316.

41±4

3.30

4 b

322.

34±4

4.95

b

DW

G (g

/day

)0.

152±

0.02

4 a

0.15

5±0.

012

a0.

149±

0.02

a0.

13±0

.018

b0.

133±

0.01

8 b

SGR

(%/d

ay)

4.15

±0.3

5 a

4.21

±0.1

6 a

4.10

8±0.

283

a3.

84±0

.283

b3.

88±0

.29

b

Bio

mas

s gai

n (g

)68

6.36

±29.

45 b

740.

12±2

2.41

a67

4.90

±18.

21 b

605.

10±1

1.95

6 c

599.

84±2

8.53

c

PER

2.00

6±0.

34 c

2.85

±0.2

2 a

2.73

±0.3

52 a

2.40

±0.3

3 b

2.44

5±0.

34 b

FCR

1.43

±0.2

5 a

1.00

8±0.

080

c1.

066±

0.15

5 c

1.21

3±0.

172

b1.

191±

0.17

b

FE (%

)70

.20±

11.8

7 c

99.8

0±7.

741

a95

.48±

12.3

12 a

84.0

0±11

.50

b85

.577

±11.

934

b

Surv

ival

rate

(%)

94.4

3±0.

4 c

98.9

7±1.

05 a

94.3

6±1.

05 c

96.1

5±0.

69 b

93.5

9±0.

79 c

som

atic

indi

ces

CF

(%)

1.85

±0.1

8 b

1.98

±0.1

1 a

1.99

±0.1

4 a

2.03

±0.1

8 a

1.98

±0.2

5 a

VSI

(%)

17.4

5±0.

87 a

15.7

5±0.

65 b

15.9

6±0.

46 b

16.0

3±0.

57 b

16.1

6±0.

67 b

HSI

(%)

3.22

±0.3

6 a

2.64

±0.3

2 b

2.69

±0.2

4 b

2.79

±0.

19 b

2.77

±0.3

3 b

CY

(%)

82.5

5±0.

87 b

84.2

5±0.

65 a

84.0

4 ±0

.46

a83

.97±

0.57

a83

.84±

0.67

a

Valu

es in

the

sam

e ro

w w

ith d

iffer

ent l

ette

rs a

re si

gnifi

cant

ly d

iffer

ent (

P<0.

05).

Bod

y w

eigh

t ind

ex =

BW

I, da

ily w

eigh

t gai

ns =

DW

G, s

peci

fic g

row

th ra

te =

SG

R, p

rote

in e

ffici

ency

ratio

= P

ER, f

eed

conv

ersi

on ra

tio =

FC

R, f

eed

effic

ienc

y =

FE.

Con

ditio

n fa

ctor

= C

F, v

isce

roso

mat

ic in

dex

= V

SI, h

epat

osom

atic

inde

x =

HSI

, car

cass

yie

ld =

CY.

BFT

24: m

olas

ses w

as a

dded

eve

ry 2

4 h.

BFT

48: m

olas

ses w

as a

dded

eve

ry 4

8 h.

BFT

72: m

olas

ses w

as a

dded

eve

ry 7

2 h.

BFT

96: m

olas

ses w

as a

dded

eve

ry 9

6 h.


Figure 1. Values of (Mean ± SD) TAN (A), NO2 (B) and NO3 (C) in tanks of Nile tilapia cultivated at different treatments during the experiment period

growth performance and somatic indicesThe values of growth and nutrition parameters during the experiment are pre-

sented in Table 3, which indicates a significant difference among some of the treat-ments (P<0.05). The growth performance was significantly lower in BFT72 and BFT96 compared to other treatments. The highest increase in biomass (740.12 g) was obtained in BFT24. The highest level of FCR (1.43) and the lowest FE (70.2%) were observed in the control group (P<0.05).

The values of the survival rate of Nile tilapia fingerlings in different treatments are presented in Table 3. The highest percentage of survival rate (98.97%) was ob-tained in BFT24. Table 3 shows the results of somatic indices including CF, VSI, HSI, and CY. According to the somatic indices, there were significant differences between BFT treatments and the control group.


Fish body proximate composition and biofloc The results of the body biochemical composition of Nile tilapia in different

treatments are demonstrated in Table 4, which shows the lowest level of protein and ash content in the control treatment. The biofloc biochemical compositions are also presented in Table 4, which show significant differences between the treatments in the amounts of ash, lipid, and protein. The highest level of protein (32.14%), lipid (2.35%) and ash (31.92%) were obtained in BFT48, BFT48 and BFT24 treatments, respectively (P<0.05).

Table 4. Values of body proximate composition of Nile tilapia (% dry weight) and biofloc obtained at the end of cultivation period in different treatments (Mean ± SD)

Treatment Dry matter (%)

Crude protein(% DW)

Crude lipid(% DW)

Ash(% DW)

fish

Control 26.16± 0.23 b 56.56± 0.32 d 25.33± 0.06 a 12.69± 0.19 c

BFT24 26.13± 0.15 b 57.17± 0.39 c 25.26± 0.07 ab 13.83± 0.10 a

BFT48 26.06± 0.12 b 57.51± 0.22 bc 25.24± 0.05 ab 13.70± 0.19 a

BFT72 26.8± 0.03 a 58.49± 0.21 a 25.16± 0.04 b 13.51± 0.12 a

BFT96 26.73± 0.18 a 57.80± 0.11 b 25.17± 0.05 b 13.11± 0.30 b

Biofloc

BFT24 18.28± 0.57 a 26.64± 0.13 d 2.17± 0.07 b 31.92± 0.22 a

BFT48 17.11± 0.26 b 32.14± 0.31 a 2.35± 0.025 a 27.39± 0.18 d

BFT72 15.76± 0.14 c 27.81± 0.33 c 2.01± 0.03 c 29.34± 0.40 b

BFT96 14.49± 0.35 d 29.89± 0.28 b 1.92± 0.15 c 28.00± 0.26 c

Values in the same column with different letters are significantly different (P<0.05).

Table 5. Digestive and hepatic enzymes activities of Nile tilapia at the end of the experiment in differ-ent treatments (Mean ± SD)

Parameters Control BFT24 BFT48 BFT72 BFT96

intestine

AM (U/mg protein) 42.52±1.5 c 46.66±2.08 b 46.27±1.73 b 53.02±2.05 a 51.87±2.20 a

LP (U/mg protein) 1.47±0.015 c 1.5±0.045 b 1.58±0.012 a 1.49±0.075 b 1.52±0.026 b

APr (U/mg protein) 9.55±0.4 c 10.74±0.53 b 12.56±0.25 a 10.69±0.53 b 11.71±0.59 b

liver

AST (U/mg protein) 2.43±0.13 b 2.03±0.09 c 2.40±0.05 b 2.71±0.13 a 2.73±0.04 a

ALT (U/mg protein) 0.4±0.02 b 0.34±0.011 c 0.37±0.01 b 0.38±0.02 b 0.42±0.009 a

ALP (U/mg protein) 0.063±0.004 a 0.046 ± 0.009 b 0.048±0.002 b 0.052±0.003 b 0.051±0.005 b

Different letters indicate significant differences in the five treatments (P<0.05).Amylase (AM), lipase (LP), alkaline protease (APr), alkaline phosphatase (ALP), alanine aminotransferase

(ALT), aspartate aminotransferase (AST).BFT24: molasses was added every 24 h. BFT48: molasses was added every 48 h. BFT72: molasses was

added every 72 h. BFT96: molasses was added every 96 h.


digestive and hepatic enzymesThe results of the analysis of factors related to digestive and hepatic enzymes

are presented in Table 5. The lowest digestive enzyme activity was obtained in the control group. The highest levels of lipase (1.58) and protease (12.56 U /mg protein) were determined in BFT48 (P<0.05). Also, the lowest activity of AST, ALT and the highest activity of ALP were observed in BFT24 and control group, respectively.

discussion

water quality Water quality including physicochemical and biological parameters are very im-

portant for maintaining the health of aquatic species and can act as a limiting factor (Kamrani et al., 2016; Yeganeh et al., 2020). According to the results, obtained phys-icochemical parameters of water are in the proper range for Nile tilapia cultivation (El-Sayed, 2006). The decrease in DO and pH is a prominent characteristic of biofloc systems (Luo et al., 2014; Khanjani et al., 2020 a, b). In the current research, the level of DO and pH were observed lower in the afternoon, probably due to the addition of organic carbonaceous molasses to the biofloc system. As microbial communities develop, carbon dioxide concentration increases due to a higher respiration rate and as a result the level of pH will reduce (Panigrahi et al., 2018; Khanjani et al., 2020 a). The DO concentration in the control group was higher than the other treatments, possibly due to the daily water exchange and the limited amount of microbial com-munities (Mirzakhani et al., 2019; Long et al., 2015). The lowest amount of DO and pH was determined in BFT24 treatment due to the higher density of heterotrophic communities.

The salinity level in the BFT treatments was higher compared to the values obtained in the control group; this is probably due to the accumulation of salts from the uneaten feed in the biofloc system (Alves et al., 2017). There is also more water evaporation in limited water exchange systems (Emerenciano et al., 2012).

In the present study, the mean values of SS and TSS in BFT treatments were consistent with the results of other studies (Avnimelech, 2012; Long et al., 2015; Lima et al., 2018). The higher level of SS (43.66 ml L–1) was measured in BFT96 treatment and the higher amount of TSS (498.8 mg L–1) in BFT24 on day 28 of the experiment, indicating that biofloc produced in BFT96 treatment is lighter and more porous than that in BFT24 treatment. It has been shown that adding molasses daily to the biofloc tanks results in a more efficient heterotrophic system, producing the microbial communities that are more compact and with more suspended solids. In the Lima et al. (2018), the level of SS was reported 13.51 to 26 ml L–1 and TSS from 45.74 to 677.17 mg L–1. The amount of TSS in biofloc-based tilapia rearing ponds can reach up to 1000 mg L–1 (Avnimelech, 2012). Too much amount of SS is not suitable for optimal fish growth, which results in increased oxygen consumption and may eventually lead to the accumulation of organic matter and closure of the fish gills (Khanjani and Sharifinia, 2020).


SS and TSS were maintained at the optimum level so as not to negatively affect growth performance. The amount of SS also impacted negatively the level of trans-parency, in a way that the amount of transparency was decreased with an increase in SS level (Khanjani et al., 2017).

Higher concentrations of TAN and NO3 were obtained in BFT treatments than the control group. The higher level of TAN is possibly due to the limited water ex-change and as a result, the accumulation of organic matter in the biofloc treatments compared to the control group. Besides, the higher level of nitrate in biofloc treat-ments could be due to the nitrification process, resulting in a higher concentration of inorganic nitrogen forms (Christopher et al., 2015; Mirzakhani et al., 2019).

The values of TAN, NO2, and NO3 in the treatments were determined in the opti-mum range of Nile tilapia cultivation, which were consistent with the results of other studies (Luo et al., 2014; Mirzakhani et al., 2019). TAN fluctuations may be due to the frequent circulation between soluble NH3 and solid flocs because of the microbial cells decomposition and the release of nitrogen in the form of NH3 (Luo et al., 2014).

The highest density of heterotrophic bacteria was obtained in BFT24 treatment and showed a significant difference with BFT96 treatment. Various studies have shown that adding molasses daily increases the density of heterotrophic bacteria dur-ing the culture period (Emerenciano et al., 2012; Khanjani et al., 2020 a).

growth performance and somatic indicesBased on the results, the growth performance in BFT24 and BFT48 was better

than BFT72 and BFT96. The highest amount of biomass was observed in BFT24 which was higher than the control group.

Studies by various researchers have reported that the biofloc system improves the growth performance of cultured aquatics (Avnimelech and Kochba, 2009; Luo et al., 2014; Wang et al., 2015; Mirzakhani et al., 2019; Khanjani et al., 2020 b). The increase in growth performance in BFT24 treatment is possibly because of maintain-ing optimal water quality and continuous and fresh production of biofloc (Ekasari et al., 2010; Toledo et al., 2016). Bioflocs contain poly-beta-hydroxybutyrate organic compounds (De Schryver et al., 2010) and bioactive compounds such as carotenoids, chlorophylls, and phytosteroids (Ju et al., 2008) that have a positive effect on the growth performance of cultured species. In the current study, the highest increase in biomass was observed in BFT24 treatment, indicating that tilapia fed better on the bioflocs that were affected daily by the addition of molasses.

The highest FCR and the lowest PFR were observed in the control group. Stud-ies have shown that the presence of biofloc with a commercial diet improves the FCR and increases feed efficiency (Khanjani et al., 2020 a; Mirzakhani et al., 2019). Bioflocs exhibit probiotic properties, which contribute to the digestion and absorp-tion of a commercial diet and lead to improved feed efficiency (Aguilera-Rivera et al., 2014).

In the present study, the highest survival rate for tilapia fingerlings was obtained in BFT24 treatment, which was significantly affected by the different addition times of molasses to the cultivation system. In the different studies of tilapia in the biofloc system, the survival rates were reported 100%, 80.4–97.6% and 67–98% by Mir-


zakhani et al. (2019), Crab et al. (2009) and Ekasari et al. (2015), respectively. The lower survival rate was observed in the BFT96 treatment which is probably because of the higher fluctuations of environmental parameters (especially pH and TAN). Fluctuations of environmental parameters such as DO, pH, deionized ammonia, NO2, and TAN caused stress to cultured aquatics and ultimately decreased growth and survival (Santacruz-Reyes and Chien, 2012; Avnimelech, 2012).

Somatic indices showed significant differences between BFT treatments and control group. The lowest levels of CF (%) and CY (%) and the highest level of VSI (%) and HSI (%) were observed in the control group. CF in the different stud-ies was reported 2.1–2.3% (Crab et al., 2009) 1.95–2.17% (Santos et al., 2016), and 1.17–1.80% (Durigon et al., 2020) for tilapia which is in the range of the results of the present study. CF level in this study was affected by the presence of biofloc, which resulted in the improvement of CF.

Feeding, age, and growth rate can be monitored by CF factor (Kumolu-Johnson and Ndimele, 2010). This factor changes with the amount of food and nutrients, so that more nutrients lead to an increase in CF levels (Morado et al., 2017).

The values of HSI, VSI, and CY were reported 2.17–2.51%, 16.21–17.44% and 82.56–83.79%, respectively (Durigon et al., 2020). The control group in the present study showed the highest level of HSI which was similar to the study of Durigon et al. (2020). They reported that the high rate of HSI is because of the compensation phenomenon in a situation of lack of access to dietary protein in which the liver size increases to metabolize muscle protein so as to meet the body’s protein requirement. Biofloc as a natural and nutritious diet that is constantly available for tilapia has a positive effect on the somatic indices (Durigon et al., 2020).

Proximate composition of fish body and bioflocIn the current study, significant differences were observed among different

treatments in the biochemical composition of Nile tilapia body and produced bio-floc, which indicates that different addition times of carbonaceous material to the cultivation tank affect the biochemical compositions of fish body and biofloc. Mir-zakhani et al (2019) reported as follows: protein 56–66%, lipid 4.5–18.6%, and ash 18–25.9% and García-Ríos et al. (2019) determined as follows: protein 63.9–71%, lipid 11.3–16.1%, ash 11.6–14.1%, for Nile tilapia in the biofloc system. The differ-ence in reported values is probably due to different experimental conditions between different studies. The lipid contents of fish cultured in the BFT treatments were lower than the control group. This is possibly because of a very low level of lipid in the biofloc (Emerenciano et al., 2012; Luo et al., 2014; Long et al., 2015; Mirzakhani et al., 2019). It has been reported that changes and higher levels of body biochemical compounds in the BFT treatments compared to control group may be due to amino acid compounds, fatty acids (PUFA, HUFA), and other nutrients found in flocs (Ju et al., 2008; Khanjani et al., 2020 a).

In the present study, the amount of protein (32.14–26.64%), lipid (2.35–1.92%) and ash (31.92–27.39%) in the flocs were obtained, which was in accordance with the reported values by Becerra-Dórame et al. (2012) and Mirzakhani et al. (2019). It was also found that adding molasses in addition times to the cultivation tanks had


a significant effect on the quality of biofloc, as the lowest ash content and the high-est protein and lipid content were measured in BFT48 treatment. These changes are probably due to differences in the composition of microbial communities and the conditions for the production of bioflocs. The lipid content of bioflocs is usually at the low level, but the presence of essential fatty acids has significantly increased their nutritional value (Ju et al., 2008; Khatoon et al., 2016). The type of carbon source (Khanjani et al., 2017), C: N ratio (Minabi et al., 2020), water salinity (Ekasari et al., 2010; Khanjani et al., 2020 b), feeding level (Khanjani et al., 2016), the source of light and its intensity (Coyle et al., 2011), pH (Martins et al., 2019) and the type of microbial community (Ahmad et al., 2017) are considered as parameters affecting the biochemical composition of bioflocs.

digestive and hepatic enzymesIn the present study, adding molasses in addition times to the cultivation tanks

affected the activity of digestive enzymes (lipase, amylase and alkaline protease) and hepatic enzymes (AST, ALT and ALP).

Digestive enzymes activity in the BFT treatments was higher than the control group, probably due to the use of biofloc and heterotrophic bacteria acting as probi-otics.

These microbial enzymes help break down proteins, carbohydrates, and other nutrients and break down the feed into smaller units and facilitate digestion and absorption (Xu and Pan, 2012). After the food was eaten with biofloc, biofloc acts as a complementary external enzyme in the diet and positively affects the function of digestive enzymes (Lin et al., 2007). Other studies have also shown that the pres-ence of exogenous bacteria can stimulate the production of endogenous enzymes by cultured fish (Ziaei-Nejad et al., 2006; Zhou et al., 2009). The activity of fish diges-tive enzymes can vary depending on the conditions of the cultivation environment, nitrogen conversion pathway (autotrophic or heterotrophic) and production system (pond or cage) (Apún-Molina et al., 2009; Becerra-Dórame et al., 2012; Santos et al., 2016; Durigon et al., 2019).

The highest amount of lipase and protease was observed in BFT48 treatment, which was affected by the amount of lipid and protein of biofloc. Amylase levels were higher in BFT72 and BFT96, which may be owing to the mixotrophic system in these treatments. The biofloc produced in these treatments is likely to have higher carbohydrates, which affects the activity of amylase.

In this study, hepatic enzymes activity was also affected by the presence of bio-floc, so the highest levels of AST, ALT and ALP were observed in BFT96 and control group, respectively, which may be due to increased anabolism or decreased catabo-lism (Shahsavani et al., 2010). Hepatic enzymes such as transaminases (AST, ALT) are used as general indicators to evaluate vertebrate liver function because increased permeability of damaged hepatocytes causes the secretion of these enzymes into the bloodstream (Suárez et al., 2015). Due to higher environmental fluctuations in BFT96 treatment, tilapia fish may have been more stressed and leads to the secretion of more liver enzymes. Chen et al. (2004) reported a significant increase in AST and ALT enzymes due to some infectious and environmental factors in Nile tilapia. The


ALT enzyme is high in the hepatocellular cytoplasm and when damaged, crosses the cell membrane and enters the bloodstream. This enzyme has been identified as a specific marker of liver injury (Hao et al., 2009). The lowest amount of this enzyme was observed in BFT24 treatment, which indicates a reduction in stress in this treat-ment and a positive effect of biofloc on the decrease in the secretion of this enzyme. Environmental stresses are factors that increase hepatic enzymes (Hao et al., 2009). Significant differences were observed in ALP enzyme levels between the biofloc treatments and control group, indicating that the presence of biofloc had a positive effect on the reduction of this hepatic enzyme.

Alkaline phosphatase is possibly related to the immune activity (Adorian et al., 2019). It has been found that physical or chemical stress or bacterial and parasitic infections increase alkaline phosphatase activity (Qi et al., 2009), which plays an important role in the immune system. Decreased activity of this enzyme in fish in biofloc treatments indicates a better immune status.

The present study showed that the addition of molasses to the biofloc system every 24 h improves the growth and development of heterotrophic bacteria, thereby reducing DO and pH levels. Biomass and survival performance of fish in BFT24 treatment were also improved compared to the other treatments. The protein and ash content of the body of Nile tilapia increased under the influence of biofloc. The activ-ity of digestive enzymes in the biofloc treatments was better than in the control group so that they were affected by the biochemical compositions of produced biofloc. It showed the lowest amount of AST and ALT activity in BFT24 treatment. Generally, it can be concluded that adding molasses every 24 h to the cultivation tanks of Nile tilapia fingerlings leads to better performance.

AcknowledgmentsWe would like to thank our coworkers, Mr. Asgari, Jafari, Hassanzadeh, De-

hghani, Karami Nasab in the National Research Center of Saline Water Aquatics (Yazd province, Iran) for their contributions in this project. We also appreciate the Honorable Research Deputy of the University of Jiroft for their collaboration in the approval and implementation of the project.

financial supportThis research was supported by University of Jiroft under the Grant NO. 3-98-

4813.

references

A d o r i a n T.J., J a m a l i H., G h a f a r i F a r s a n i H., D a r v i s h i P., H a s a n p o u r S., B a g - h e r i T., R o o z b e h f a r R. (2019). Effects of probiotic bacteria bacillus on growth performance, digestive enzyme activity, and hematological parameters of Asian sea bass, Lates calcarifer (Bloch). Probiotics Antimicrob. Proteins, 11: 248–255.

A g u i l e r a - R i v e r a D., P r i e t o - D a v ó A., E s c a l a n t e K., C h á v e z C., C u z o n G., G a x i o - l a G. (2014). Probiotic effect of FLOC on Vibrios in the pacific white shrimp Litopenaeus vanna-mei. Aquaculture, 424: 215–219.


A h m a d I., B a b i t h a R a n i A.M., Ve r m a A.K., M a q s o o d M. (2017). Biofloc technology: an emerging avenue in aquatic animal healthcare and nutrition. Aquacult. Int., 25: 1215–1226.

A l v e s G.F.O., F e r n a n d e s A.F.A., A l v a r e n g a E.R., T u r r a E.M., S o u s a A.B., T e i x e i - r a E.A. (2017). Effect of the transfer at different moments of juvenile Nile tilapia (Oreochromis niloticus) to the biofloc system in formation. Aquaculture, 479: 564–570.

AOAC (2005). Official methods of analysis. Association of Official Analytical Chemists, INC., Arling-ton, Virginia, USA, p. 245.

APHA (2005). American Water Works Association, Water Pollution Control Association. Standard Methods for the Examination of Water and Wastewater (21st ed.). American Public Health Associa-tion, Washington, DC, USA.

A p ú n - M o l i n a J.P., S a n t a m a r í a - M i r a n d a A., L u n a - G o n z á l e z A., M a r t í n e z - - D í a z S.F., R o j a s - C o n t r e r a s M. (2009). Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimum temperature. Aquac. Res., 40: 887–894.

A v n i m e l e c h Y. (2007). Feeding with microbial flocs by tilapia in minimal discharge bio-flocs tech-nology ponds. Aquaculture, 264: 140–147.

A v n i m e l e c h Y. (2009). Biofloc Technology – A Practical Guide Book. 1st ed. The World Aquacul-ture Society, Baton Rouge, LA, USA, 182 pp.

A v n i m e l e c h Y. (2012). Biofloc Technology – A Practical Guide Book. 2nd ed. The World Aquacul-ture Society, Baton Rouge, USA, 272 pp.

A v n i m e l e c h Y., K o c h b a M. (2009). Evaluation of nitrogen uptake and excretion by tilapia in bio floc tanks, using 15N tracing. Aquaculture, 287: 163–168.

B a k h s h i F., N a j d e g e r a m i E.H., M a n a f f a r R., T o k m e c h i A., F a r a h K.R., J a l a l i A.S. (2018). Growth performance, haematology, antioxidant status, immune response and histology of common carp (Cyprinus carpio L.) fed biofloc grown on different carbon sources. Aquac. Res., 49: 393–403.

B e c e r r a - D ó r a m e M., M a r t i n e z - P o r c h a s M., M a r t i n e z - C o r d o v a L.R., R i v a s - - Ve g a M.E., L o p e z - E l i a s J.A., P o r c h a s - C o r n e j o M.A. (2012). Production response and digestive enzymatic activity of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) intensively pre grown in microbial heterotrophic and autotrophic-based systems. Sci. World J., 723654, 6 pp.

B e r g m e y e r H.U., H o r d e r M., R e j R. (1986). International Federation of Clinical Chemistry (IFCC) Scientific Committee. J. Clin. Chem. Clin. Biochem., 24: 497–510.

B e r n f e l d P. (1955). Amylase. In: Methods in Enzymology, Colowick S.P., Kaplan N.O. (eds.). Aca-demic Press, New York, pp: 149–158.

C h e n C., Wo o s t e r G.A., B o w s e r P.R. (2004). Comparative blood chemistry and histopathology of tilapia infected with Vibrio vulnificus or Streptococcus iniae or exposed to carbon tetrachloride, gentamicin or copper sulfate. Aquaculture, 239: 421–443.

C h r i s t o p h e r M.A., C a i p a n g H.X., C h o o Z.B., H u i l i n H., C l a r a M., L a y - Ya g J.L. (2015). Small-scale production of biofloc using various carbon sources for the freshwater culture of tilapia, Oreochromis sp. ABAH Bioflux, 7: 103–111.

C o l t J. (2006). Water quality requirements for reuse systems. Aquac. Eng., 34: 143–156. C o y l e S.D., B r i g h t L.A., Wo o d D.R., N e a l R.S., T i d w e l l J.H. (2011). Performance of Pacific

white shrimp, Litopenaeus vannamei, reared in zero-exchange tank systems exposed to different light sources and intensities. J. World Aquacult. Soc., 42: 687–695.

C r a b R., K o c h v a M., Ve r s t r a e t e W., A v n i m e l e c h Y. (2009). Bio-flocs technology applica-tion in over-wintering of tilapia. Aquac. Eng., 40: 105–112.

D e S c h r y v e r P., S i n h a A.K., K u n w a r P.S., B a r u a h K., Ve r s t r a e t e W., B o o n N., D e B o e c k G., B o s s i e r P. (2010). Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax. Appl. Microbiol. Biotechnol., 86: 1535–1541.

D e n g M., C h e n J., G o u J., H o u J., L i D., H e X. (2018). The effect of different carbon sources on water quality, microbial community and structure of biofloc systems. Aquaculture, 482: 103–110.

Deutsche Gesellschaft für Klinische Chemie (1972). Empfehlungen der deutschen Gesellschaft für Klinische Chemie. Standardisierung von Methoden zur Bestimmung von Enzymaktivitaten in bi-


ologischen flussigkeiten. (Standardizition of methods for measurement of enzymatic activities in biological fluids). Z. Klin. Chem. Klin. Biochem., 10: 182–192.

D u r i g o n E.G., A l m e i d a A.P.G., J e r ô n i m o G.T., B a l d i s s e r o t t o B., E m e r e n c i a - n o a M.G.C. (2019). Digestive enzymes and parasitology of Nile tilapia juveniles raised in brack-ish biofloc water and fed with different digestible protein and digestible energy levels. Aquaculture, 506: 35–41.

D u r i g o n E.G., L a z z a r i R., U c z a y J., L o p e s D.L.D.A., J e r ô n i m o G.T., S g n a u l i n T., E m e r e n c i a n o M.G.C. (2020). Biofloc technology (BFT): Adjusting the levels of digestible protein and digestible energy in diets of Nile tilapia juveniles raised in brackish water. Aquacult. Fish., 5: 42–51.

E k a s a r i J., C r a b R., Ve r s t r a e t e W. (2010). Primary nutritional content of bio-flocs cultured with different organic carbon sources and salinity. HAYATI J. Biosci., 17: 125–130.

E k a s a r i J., R i v a n d i D.R., F i r d a u s i A.P., S u r a w i d j a j a E.H., Z a i r i n M., B o s s i e r P., D e S c h r y v e r P. (2015). Biofloc technology positively affects Nile tilapia (Oreochromis niloti-cus) larvae performance. Aquaculture, 441: 72–77.

E l - S a y e d E. M. (2006). Tilapia Culture. CABI Publishing, Cambridge Massachusetts, USA, 275 p.E m e r e n c i a n o M., B a l l e s t e r E.L., C a v a l l i R.O., Wa s i e l e s k y W. (2012). Biofloc tech-

nology application as a food source in a limited water exchange nursery system for pink shrimp Farfantepenaeus brasiliensis (Latreille, 1817). Aquac. Res., 43: 447–457.

E m e r e n c i a n o M.G.C., M a r t í n e z - C ó r d o v a L.R., M a r t í n e z - P o r c h a s M., M i r a n -d a - B a e z a A. (2017). Biofloc technology (BFT): a tool for water quality management in aqua-culture. Water Quality, InTech, London, UK, pp. 91–109.

G a r c i a - C a r r e n o F.L., H a a r d N.F. (1993). Characterization of proteinase classes in langostilla (Pleuroncodes planipes) and crayfish (Pacifastacus astacus) extracts. J. Food Biochem., 17: 97–113.

G a r c í a - R í o s L., M i r a n d a - B a e z a A., C o e l h o - E m e r e n c i a n o M.G., H u e r t a - - R á b a g o J.A., O s u n a - A m a r i l l a s P. (2019). Biofloc technology (BFT) applied to tilapia fingerlings production using different carbon sources: Emphasis on commercial applications. Aqua-culture, 502: 26–31.

H a k i m Y., U n i Z., H u l a t a G., H a r p a z S. (2006). Relationship between intestinal brush border enzymatic activity and growth rate in tilapias fed diets containing 30% or 48% protein. Aquaculture, 257: 420–428.

H a o L., Wa n g Z., X i n g B. (2009). Effect of sub-acute exposure to TiO nanoparticles on oxida-tive stress and histopathological changes in juvenile carp (Cyprinus carpio). J. Environ. Sci., 21: 1459–1466.

H a r i d a s H., Ve r m a A.K., R a t h o r e G., P r a k a s h C., B a n e r j e e P. (2017). Enhanced growth and immuno-physiological response of Genetically Improved Farmed Tilapia in indoor biofloc units at different stocking densities. Aquac. Res., 48: 4346–4355.

H u Z., L e e J.W., C h a n d r a n K., K i m S., B r o t t o A.C., K h a n a l S.K. (2015). Effect of plant species on nitrogen recovery in aquaponics. Bioresour. Technol., 188: 92–98.

I i j i m a N., T a n a k a S., O t a Y. (1998). Purification and characterization of bile salt-activated lipase from the hepatopancreas of red sea bream (Pagrus major). Fish Physiol. Biochem., 18: 59–69.

J e n a b i H a g h p a r a s t R., M o g h a n l o u K.S., M o h s e n i M., I m a n i A. (2019). Effect of dietary soybean lecithin on fish performance, hemato-immunological parameters, lipid biochemis-try, antioxidant status, digestive enzymes activity and intestinal histomorphometry of pre-spawning Caspian brown trout (Salmo trutta caspius). Fish Shellfish Immunol., 91: 50–57.

J u Z., F o r s t e r I., C o n q u e s t L., D o m i n y W. (2008). Enhanced growth effects on shrimp (Lito-penaeus vannamei) from inclusion of whole shrimp floc or floc fractions to a formulated diet. Aquac. Nutr., 14: 533–543.

K a m r a n i E., S h a r i f i n i a M., H a s h e m i S.H. (2016). Analyses of fish community structure changes in three subtropical estuaries from the Iranian coastal waters. Mar. Biodivers., 46: 561–577.

K h a n j a n i M.H., S h a r i f i n i a M. (2020). Biofloc technology as a promising tool to improve aqua-culture production. Rev. Aquacult., 12: 1836–1850.

K h a n j a n i M.H., S a j j a d i M., A l i z a d e h M., S o u r i n e j a d I. (2016). Study on nursery growth performance of Pacific white shrimp (Litopenaeus vannamei Boone, 1931) under different feeding levels in zero water exchange system. Iran. J. Fish. Sci.,15: 1465–1484.


K h a n j a n i M.H., S a j j a d i M.M., A l i z a d e h M., S o u r i n e j a d I. (2017). Nursery performance of Pacific white shrimp (Litopenaeus vannamei Boone, 1931) cultivated in a biofloc system: the ef-fect of adding different carbon sources. Aquac. Res., 48: 1491–1501.

K h a n j a n i M.H., A l i z a d e h M., S h a r i f i n i a M. (2020 a). Rearing of the Pacific white shrimp, Litopenaeus vannamei in a biofloc system: The effects of different food sources and salinity levels. Aquac. Nutr., 26: 328–337.

K h a n j a n i M.H., S h a r i f i n i a M. H a j i r e z a e e S. (2020 b). Effects of different salinity levels on water quality, growth performance and body composition of Pacific white shrimp (Litopenaeus vannamei Boone, 1931) cultured in a zero water exchange heterotrophic system. Ann. Anim. Sci., 20: 1–16.

K h a n j a n i M.H., A l i z a d e h M., S h a r i f i n i a M. (2021 a). Effects of different carbon sources on water quality, biofloc quality, and growth performance of Nile tilapia (Oreochromis niloticus) fingerlings in a heterotrophic culture system. Aquacult. Int., 29: 307–321.

K h a n j a n i M.H., A l i z a d e h M., M o h a m m a d i M., S a r s a n g i A l i a b a d H. (2021 b). Bio-floc system applied to Nile tilapia (Oreochromis niloticus) farming using different carbon sources: growth performance, carcass analysis, digestive and hepatic enzyme activity. Iran. J. Fish. Sci., 20: 490–513.

K h a t o o n H., B a n e r j e e S., Yu a n G., H a r i s N., I k h w a n u d d i n M., A m b a k M., E n d u -t e t A. (2016). Biofloc as a potential natural feed for shrimp postlarvae. Int. Biodeterior. Biodegrad., 113: 304–309.

K u m o l u - J o h n s o n C.A., N d i m e l e P.E. (2010). Length-weight relationships and condi-tion factors of twenty-one fish species in Ologe Lagoon, Lagos, Nigeria. Asian J. Agric. Sci., 4: 174–179.

L i m a P.C.M., A b r e u J.L., S i l v a A.E.M., S e v e r i W., G a l v e z A.O., B r i t o L.O. (2018). Nile tilapia fingerling cultivated in a low-salinity biofloc system at different stocking densities. Span. J. Agric. Res., 16: 612–621.

L i n S., M a i K., T a n B. (2007). Effects of exogenous enzyme supplementation in diets on growth and feed utilization in tilapia, Oreochromis niloticus×O. aureus. Aquac. Res., 38: 1645–1653.

L i u G., Ye Z., L i u D., Z h a o J., S i v a r a m a s a m y E., D e n g Y., Z h u S. (2018). Influence of stocking density on growth, digestive enzyme activities, immune responses, antioxidant of Oreo-chromis niloticus fingerlings in biofloc systems, Fish Shellfish Immunol., 81: 416–422.

L o n g L., Ya n g J., L i Y., G u a n C., W u F. (2015). Effect of biofloc technology on growth, digestive enzyme activity, hematology, and immune response of genetically improved farmed tilapia (Oreo-chromis niloticus). Aquaculture, 448: 135–141.

L u o G., Wa n g C., L i u W., S u n D., L i L., T a n H. (2014). Growth, digestive activity, welfare, and partial cost-effectiveness of genetically improved farmed tilapia (Oreochromis niloticus) cultured in a recirculating aquaculture system and an indoor biofloc system. Aquaculture, 422–423: 1–7.

M á r q u e z A.G., D e m e s s e n c e A., P l a t e r o - P r a t s A.E., H e u r t a u D., H o r c a j a d a P., S e r r e C., C h a n g J.S., F é r e y G., d e l a P e ñ a - O ' S h e a V.A., B o i s s i è r e C., G r o s - s o D., S a n c h e z C. (2012). Green microwave synthesis of MIL-100 (Al, Cr, Fe) nanoparticles for thin-film elaboration. Eur. J. Inorg. Chem., 100: 5165–5174.

M a r t i n s G.B., d a R o s a C.E., T a r o u c o F.M, R o b a l d o R.B. (2019). Growth, water quality and oxidative stress of Nile tilapia Oreochromis niloticus (L.) in biofloc technology system at different pH. Aquac. Res., 50: 1030–1039.

M e n a g a M., F e l i x b S., C h a r u l a t h a M., G o p a l a k a n n a n a A., P a n i g r a h i c A. (2019). Effect of in-situ and ex-situ biofloc on immune response of Genetically Improved Farmed Tilapia. Fish Shellfish Immunol., 92: 698–705.

M i n a b i K., S o u r i n e j a d I., A l i z a d e h M., R a j a b z a d e h G h a t r a m i E., K h a n j a - n i M.H. (2020). Effects of different carbon to nitrogen ratios in the biofloc system on water quality, growth, and body composition of common carp (Cyprinus carpio L.) fingerlings. Aquacult. Int., 28: 1883–1898.

M i r z a k h a n i N., E b r a h i m i E., J a l a l i S.A.H., E k a s a r i J. (2019). Growth performance, in-testinal morphology and nonspecific immunity response of Nile tilapia (Oreochromis niloticus) fry cultured in biofloc systems with different carbon sources and input C:N ratios. Aquaculture, 512: 734235.


MOOPAM (1999). Manual of oceanographic observations and pollutant analysis methods. Kuwait, ROPME, 1: 20.

M o r a d o C.N., A r a ú j o F.G., G o m e s I.D. (2017). The use of biomarkers for assessing effects of pollutant stress on fish species from a tropical river in Southeastern Brazil. Acta Sci., 39: 431–439.

N a j d e g e r a m i E.H., B a k h s h i F., L a k a n i F.B. (2016). Effects of biofloc on growth perfor-mance, digestive enzyme activities and liver histology of common carp (Cyprinus carpio L.) finger-lings in zero-water exchange system. Fish Physiol. Biochem., 42: 457–465.

P a n i g r a h i A., S a r a n y a C., S u n d a r a m M., K a n n a n S.V., D a s R.R., K u m a r R.S., R a j e s h P., O t t a S. (2018). Carbon: Nitrogen (C: N) ratio level variation influences microbial community of the system and growth as well as immunity of shrimp (Litopenaeus vannamei) in biofloc based culture system. Fish Shellfish Immunol., 81: 329–337.

P é r e z - F u e n t e s J.A., H e r n á n d e z - Ve r g a r a M.P., P é r e z - R o s t r o C.I., F o g e l I. (2016). C:N ratios affect nitrogen removal and production of Nile tilapia Oreochromis niloticus raised in a biofloc system under high density cultivation. Aquaculture, 452: 247–251.

P i n h o S.M., M o l i n a r i D., d e M e l l o G.L., F i t z s i m m o n s K.M., E m e r e n c i a n o M.G.C. (2017). Effluent from a biofloc technology (BFT) tilapia culture on the aquaponics production of different lettuce varieties. Ecol. Eng., 103: 146–153.

Q i Z., Z h a n g X.H., B o o n N., B o s s i e r P. (2009). Probiotics in aquaculture of China – current state, problems and prospect. Aquaculture, 290: 15–21.

R e n W., L i L., D o n g S., T i a n X., X u e Y. (2019). Effects of C/N ratio and light on ammonia nitro-gen uptake in Litopenaeus vannamei culture tanks. Aquaculture, 498: 123–131.

S a n t a c r u z - R e y e s R.A., C h i e n Y.H. (2012). The potential of Yucca schidigera extract to reduce the ammonia pollution from shrimp farming. Bioresour. Technol., 113: 311–314.

S a n t o s J.F., S o a r e s K.L.S., A s s i s C.R.D., G u e r r a C.A.M., L e m o s D., C a r v a l h o L.B., B e z e r r a R.S. (2016). Digestive enzyme activity in the intestine of Nile tilapia (Oreochromis niloticus L.) under pond and cage farming systems. Fish Physiol. Biochem., 42: 1259–1274.

S e i x a s F i l h o J.T., O l i v e i r a M.G.A., D o n z e l e J.L., G o m i d e A.T.M., M e n i n E. (2000). Lipase activity in the chime of three Teleostei freshwater fish. Rev. Bras. Zootec., 29: 6–14.

S h a h s a v a n i D., K a z e r a n i H.R., K a v e h S., G h o l i p o u r - K a n a n i H. (2010). Determina-tion of some normal serum parameters in starry sturgeon (Acipenser stellatus Pallas, 1771) during spring season. Comp. Clin. Path., 19: 57–61.

S u á r e z M.D., T r e n z a d o C.E., G a r c í a - G a l l e g o M., F u r n é M., G a r c í a - M e s a S., D o m e z a i n A., A l b a I., S a n z A. (2015). Interaction of dietary energy levels and culture den-sity on growth performance and metabolic and oxidative status of rainbow trout (Oncorhynchus mykiss). Aquac. Eng., 67: 59–66.

T o l e d o T.M., S i l v a B.C., V i e i r a F.D.N., M o u r i n o J.L.P., S e i f f e r t W.Q. (2016). Effects of different dietary lipid levels and fatty acids profile in the culture of white shrimp Litopenaeus van-namei (Boone) in biofloc technology: water quality, biofloc composition, growth and health. Aquac. Res., 47: 1841–1851.

Wa n g G., Yu E., X i e J., Yu D., L i Z., L u o W., Q i u L., Z h e n g Z. (2015). Effect of C:N ratio on water quality in zero-water exchange tanks and the biofloc supplementation in feed on the growth performance of crucian carp, Carassius auratus. Aquaculture, 443: 98–104.

Wa n g M., L u M. (2016). Tilapia polyculture: a global review. Aquac. Res., 47: 2363–2374.X u W.J., P a n L.Q. (2012). Effects of bioflocs on growth performance, digestive enzyme activity and

body composition of juvenile Litopenaeus vannamei in zero-water exchange tanks manipulating C/N ratio in feed. Aquaculture, 356: 147–152.

X u W.J., P a n L.Q. (2014). Dietary protein level and C/N ratio manipulation in zero exchange culture of Litopenaeus vannamei: Evaluation of inorganic nitrogen control, biofloc composition and shrimp performance. Aquac. Res., 45: 1842–1851.

Ye g a n e h V., S h a r i f i n i a M., M o b a r a k i S., D a s h t i a n n a s a b A., A e i n j a m s h i d K., B o r a z j a n i J.M., M a g h s o u d l o o T. (2020). Survey of survival rate and histological altera-tions of gills and hepatopancreas of the Litopenaeus vannamei juveniles caused by exposure of Margalefidinium / Cochlodinium polykrikoides isolated from the Persian Gulf. Harmful Algae, 97: 101856.


Z h o u X.X., Wa n g Y.B., L i W.F. (2009). Effect of probiotic on larvae shrimp (Penaeus vannamei) based on water quality, survival rate and digestive enzyme activities. Aquaculture, 287: 349–353.

Z i a e i - N e j a d S., R e z a e i M.H., T a k a m i G.A., L o v e t t D.L., M i r v a g h e f i A.R., S h a k o - u r i M. (2006). The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activ-ity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquaculture, 252: 516–524.

Received: 26 IX 2020Accepted: 28 I 2021

ann. anim. sci., vol. 21, no. 4 (2021) 1435–1454 doi: 10

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